Blood platelets are small anucleate cells that are crucial for the arrest of bleeding. There are many clinical diseases where platelet production or function is impaired and the number of patients who require platelet transfusion is increasing. Presently, the solution consists in conducting transfusions of platelets obtained from blood donors. Ex vivo platelet production for therapeutic applications is an appealing alternative, but remains a major technological challenge.
Platelets originate from megakaryocytes. Megakaryocyte differentiation is a continuous process characterized by sequential steps. While megakaryocyte differentiation takes place in the bone marrow, platelet production requires the passage of megakaryocyte fragments into the vessels of the bone marrow. Attempts of designing bioreactors specifically dedicated to platelet production have been made.
The international patent application WO 2010/06382311 discloses an ex vivo method for producing platelets from mature megakaryocytes by subjecting a suspension of mature megakaryocytes to a flow having a shear rate of at least 600 s−1 on a solid phase coated with von Willebrand factor (VWF). The shear rate influence is further discussed in the scientific article of Dunois-Lardé et al. (“Exposure of human megakaryocytes to high shear rates accelerates platelet production”, Blood, vol. 114, no 9, p. 1875-1883, 2009). However, the production yield still needs to be improved for potential therapeutic applications.
Some publications suggest the use of 3D systems designed to reproduce the natural bone marrow environment (see Pallotta et al, “Three-Dimensional System for the In Vitro Study of Megakaryocytes and Functional Platelet Production Using Silk-Based Vascular Tubes”, Tissue Engineering: Part C, vol. 17, no 12, p. 1223-1232, 2011, and Sullenbarger et al., “Prolonged continuous in vitro human platelet production using three-dimensional scaffolds”, Experimental Hematology, vol. 37, no 1, p. 101-110, 2009). In these systems, cells fragments freely migrate from a scaffold in which cells are embedded into a flowing channel through porosity or tubes. In addition, Nakagawa et al (“Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes”, Experimental Hematology, 2013 vol. 41, no 8 p 742-748, 2013) recently disclose two new bioreactors which mimic a capillary blood vessel for platelet production. They comprise a porous structure or slits able to trap megakaryocytes. A pressure flow is applied to ensure the fixation of the megakaryocytes. In addition, a main flow is applied perpendicular to the pressure flow or with an angle of 60° between pressure flow and main flow. Said main flow applies shear stress to the trapped megakaryocytes. However, the main flow remains free of megakaryocytes. Megakaryocyte fragments are subjected to the main flow and released platelets are collected at the outlet of the main flow. Thon et al (<<Platelet bioreactor-on-a-chip>>, Blood, vol 124, no 12 p 1857-1867, 2014) describe another bioreactor based on a feeding channel and main channel flow parallel to one another, separated by a wall pierced with slits. When the end of the feeding channel is closed, megakaryocytes are pushed through the slits. There, they are brought into contact with the main flow and experience shear stress. In such systems, the number of sites available for megakaryocytes is limited, and many cells stay stuck in the reservoir while the first ones elongate and release platelets. This limits the speed of the platelet production process. These system geometries are not efficient for rapid large-scale production. In addition, the obtained production yields are still not sufficient, a fact that renders difficult the functional characterization of platelets. Furthermore, experiments performed over several hours or days have an intrinsic platelet shedding that is seldom evaluated.
Microfluidic devices with specific structure have been disclosed for different biological applications. For instance, Stott et al. (“Isolation of circulating tumor cells using a microvortex-generating herringbone-chip”, PNAS, vol. 107, no 43, p. 18392-18397, 2010) describe the use of a microfluidic device whose inner surface is coated with antibodies for isolation of circulating tumor cells. Grooves were formed in the inner surface, so as to disrupt the laminar flow streamlines inside the channel, and to increase the number of cell-surface interactions in the antibody-coated device. Similarly, Chang et al. (“Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel”, Lab Chip, vol. 5, p. 64-73, 2005) teach the use of a microfluidic channel containing arrays of micropillars to separate and collect cells. However, contrary to the present invention, the object of such microfluidic device is not to trigger platelet shedding from megakaryocytes.
One object of the present invention is therefore to provide a fluidic device for producing platelets from a suspension of cells comprising megakaryocytes or their fragments, in particular suitable for high yield production while maintaining the functional qualities of the newly generated platelets. Another object of the invention is to provide a method for producing platelets suitable to large-scale production of high quality and standardized platelets.